EP3015865A1 - Biomarker für kognitiver dysfunktionserkrankungen und verfahren zur erkennung von kognitiven dysfunktionserkrankungen unter verwendung von biomarkern - Google Patents

Biomarker für kognitiver dysfunktionserkrankungen und verfahren zur erkennung von kognitiven dysfunktionserkrankungen unter verwendung von biomarkern Download PDF

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EP3015865A1
EP3015865A1 EP13888380.6A EP13888380A EP3015865A1 EP 3015865 A1 EP3015865 A1 EP 3015865A1 EP 13888380 A EP13888380 A EP 13888380A EP 3015865 A1 EP3015865 A1 EP 3015865A1
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cognitive dysfunction
biomarker
dysfunction diseases
biomarkers
detecting
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French (fr)
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EP3015865B1 (de
EP3015865A4 (de
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Kazuhiko Uchida
Kohji Meno
Hideaki Suzuki
Yoshinori Nishimura
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University of Tsukuba NUC
MCBI Inc
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University of Tsukuba NUC
MCBI Inc
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896Neurological disorders, e.g. Alzheimer's disease
    • GPHYSICS
    • G06COMPUTING; CALCULATING OR COUNTING
    • G06FELECTRIC DIGITAL DATA PROCESSING
    • G06F17/00Digital computing or data processing equipment or methods, specially adapted for specific functions
    • G06F17/10Complex mathematical operations
    • G06F17/18Complex mathematical operations for evaluating statistical data, e.g. average values, frequency distributions, probability functions, regression analysis
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/16Aptamers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775Apolipopeptides
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/948Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433Serine endopeptidases (3.4.21)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/28Neurological disorders
    • G01N2800/2814Dementia; Cognitive disorders
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/28Neurological disorders
    • G01N2800/2814Dementia; Cognitive disorders
    • G01N2800/2821Alzheimer

Definitions

  • the present invention relates to biomarkers which are novel proteins and peptides capable of being employed for detecting cognitive dysfunction diseases including mild cognitive impairment and Alzheimer's disease, and methods for detecting cognitive dysfunction diseases using the biomarkers.
  • a major prior art as a means for using samples exhibiting in vivo conditions which are normal and are not normal for determining a difference between them is a technology employed generally in extracorporeal diagnostic agents.
  • the prior art in this field involves blood tests to quantify a single certain protein or oligopeptide having a molecular weight of 10,000 or less, or to determine the activity in cases of enzymatic proteins, thereby identifying a marked difference between the normal sample (healthy individual) and the disease sample, which serves as an aid in the diagnosis.
  • a certain number of biological samples from healthy and diseased individuals are assayed for levels or activities of a single or multiple certain proteins or certain oligopeptides to determine the abnormal and normal ranges. Then, a biological sample to be evaluated is measured by the same method and the evaluation is made based on the range, abnormal or normal, in which the measured value is included.
  • Typical measurement methods include an enzyme linked immmunosorbent assay (ELISA) in which the levels of a single or multiple certain proteins or peptides in a sample which has been or has not been diluted preliminarily is measured based on the colorimetric level of the sample using a specific primary antibody or secondary antibody labeled with an enzyme which develops a color when reacting with a substrate, as well as a chemiluminescent immunoassay (CLIA).
  • ELISA enzyme linked immmunosorbent assay
  • RIA radioimmunoassay
  • HPLC high pressure liquid chromatography
  • SRM selected reaction monitoring
  • MRM multiple reaction monitoring
  • a sample is pretreated appropriately and then subjected to a two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate proteins or peptides, and thereafter the intended protein or peptide is subjected to a silver staining, coomassie blue staining, or immunostaining employing a relevant antibody (western blotting), thereby measuring the concentration in the sample.
  • 2D-PAGE two-dimensional polyacrylamide gel electrophoresis
  • a biological sample is fractionated by a column chromatography and a fraction is subjected to a mass spectrometry to analyze the proteins and the peptides contained therein.
  • a pretreatment using a protein chip is conducted for a mass spectrometry, or a pretreatment using magnetic beads is conducted for a mass spectrometry.
  • the inventor also had developed an immunoMS method in which beads (including magnetic beads) are allowed to bind to an antibody against the target protein or peptide, thereby capturing the target protein or peptide which are thereafter allowed to elute from the beads and measured by the mass spectrometry.
  • a direct fractionation is conducted utilizing the nature of an intact protein, or a specifically adsorbing protein molecule is selected for mass spectrometric analysis.
  • Cognitive dysfunction diseases a representative of which is Alzheimer's disease, are prevailing rapidly also in Japan in response to an increase in the population of the aged.
  • the population was about 1,300,000 in 1995, and then increased to about 2,800,000 in 2010, and is assumed to approximate 4,100,000 in 2020.
  • the Alzheimer's disease is believed to account for 60 to 90% of the cognitive dysfunction diseases. This disease is becoming a social problem because it makes a patient suffer not only from loss of memory but also from destruction of personality, thereby impairing the social life function of the patient.
  • donepezil hydrochloride which is an anti-acetylcholine esterase inhibitor
  • an early diagnosis is a greatest challenge for achieving the effectiveness of current therapeutic methods or drugs which will be developed in future.
  • DSM IV Alzheimer's disease
  • AD Alzheimer's disease
  • precursory condition of the cognitive disease a condition which should be referred to as precursory condition of the cognitive disease.
  • MCI mild cognitive impairment
  • MCI is regarded as a condition in which complaint relating to a reduction in the cognitive functions is given but there is no particular difficulty in everyday life.
  • Frontotemporal dementia (FTD) is characterized by reduced cognitive functions together with a selfish behavior regardless of the circumstance, and is in contrast with AD in which a patient makes an effort to be in harmony with the circumstance.
  • FTD includes Pick's disease in which a pick body is observed histologically in cerebral cortex.
  • Dementia with Lewy Bodies is characterized by progressive memory disturbance and visual cognitive impairment such as hallucination. Based on the diagnosis of clinical signs, 10 to 30% of cognitive disease is DLB, whose morbidity is second highest after Alzheimer-type cognitive disease (AD) among senile degenerative cognitive disease types. Histologically, Lewy Body is present characteristically in cerebral cortex. Since FTD and DLB exhibit a cognitive disease and are of a dementia type, they are referred to also as dementia-type neurological diseases (Non-Patent Document 1).
  • HDS-R Revised Hasegawa's Dementia Scale
  • MMSE Mini-Mental State Examination
  • Image-based diagnostic methods are CT/MRI which observes morphological abnormality in brain such as encephalatrophy and enlarged vadum and ventricle, single photon emission computed tomography (SPECT) which measures cerebral blood flow level, and positron emission tomography (PET) which measures oxygen consumption/glucose consumption.
  • SPECT and PET are a nuclear medicine-based methods and believed to be capable of detecting abnormality before onset of morphological abnormality (Non-Patent Document 1). Nevertheless, these image-based diagnostic methods have drawbacks due to difficulty in being conducted in every medical facility because of special devices required for them. In addition, they are not sufficient for give an objective decision because the decision differs from physician to physician who observes the image.
  • the diagnosis of the cognitive disease depends currently on a method which is less objective and requires expensive instruments, and is not successful in screening for identifying the disease. If a biomarker which enables an objective diagnosis using a readily obtainable patient's sample such as blood (including serum and plasma) is found here, a screening can be conducted, thereby enabling an early detection of a cognitive dysfunction disease which is the mest challenging in these days.
  • Patent Document 1 discloses a method for detecting a change in the level of apolipoprotein A-II in a serum of a cancer-carrying mammal in which the quantified levels of the apolipoprotein A-II contained in multiple serum samples derived from the aforementioned identical cancer-carrying mammal are compared with each other among the aforementioned samples.
  • Patent Document 2 discloses a hepatoma biomarker for detecting a hepatoma consisting of at least one protein or peptide selected from the group consisting of, for example, fibrinogen ⁇ chain consisting of the amino acid sequence represented by SEQ ID NO:1 described in this relevant Document 2.
  • Patent Documents 3 to 5 disclose biomarkers for diagnosis of cognitive dysfunction diseases.
  • Patent Document 5 discloses a biomarker for detecting cognitive dysfunction diseases, which consists of at least one protein or peptide selected from, for example, complement C3 consisting of the amino acid sequence represented by SEQ ID NO:1 described in this relevant Document 5, transthyretin consisting of the amino acid sequence represented by SEQ ID NO:15 described in this relevant Document 5, or a peptide fragment having 5 or more amino acid residues from the aforementioned protein or peptide.
  • An object of the present technology is to provide a biomarker and a method for detecting a cognitive dysfunction disease using the biomarker.
  • the inventor has searched for proteins and their partial peptides which are present or absent or are present in different levels in non-demented control subjects and patients having cognitive dysfunction diseases.
  • polypeptides capable of detecting cognitive dysfunction diseases in serum. These three polypeptides were identified to be (a) a peptide derived from apolipoprotein A1 consisting of the amino acid sequence represented by SEQ ID NO:1, (b) a peptide derived from transthyretin consisting of the amino acid sequence represented by SEQ ID NO:2, and (c) a peptide derived from complement C3 consisting of the amino acid sequence represented by SEQ ID NO:3.
  • the inventor also analyzed the combinations of the biomarkers which exhibited high percentages of correct answers in detecting the cognitive dysfunction diseases using a logistic regression analysis.
  • the combination of two peptides namely, the aforementioned (c) a peptide derived from complement C3 and the aforementioned (b) a peptide derived from transthyretin or the aforementioned (a) a peptide derived from apolipoprotein A1 can achieve a higher detection accuracy for the cognitive dysfunction diseases (especially mild cognitive dysfunction and Alzheimer's disease).
  • the non-demented control (NDC) subject includes healthy humans, and means the subjects which may have any diseases but not have cognitive dysfunction diseases (non-demented control).
  • MCI mild cognitive impairment
  • AD Alzheimer's disease
  • AD dementia-type neurological diseases
  • mild cognitive impairment (MCI) and AD can preferably be detected.
  • the peptides identified in the present technology have significance as biomarkers when detected not only in serum but also in other biological samples such as blood, serum, plasma, cerebrospinal fluid, urine, and the like.
  • source proteins from which the aforementioned peptides are derived hereinafter referred to as "intact proteins" or source peptides also have a significance as biomarkers.
  • the present technology provides one or more biomarkers for detecting cognitive dysfunction diseases selected from the following (a), (b), and (c):
  • the present technology provides a method for detecting cognitive dysfunction diseases in which one or more biomarkers for detecting cognitive dysfunction diseases selected from the aforementioned (a) biomarker, (b) biomarker, and (c) biomarker in a biological sample are measured simultaneously or separately.
  • the present technology provides a kit for detecting cognitive dysfunction diseases for measuring one or more biomarkers for detecting cognitive dysfunction diseases selected from the aforementioned (a) biomarker, (b) biomarker, and (c) biomarker.
  • the present technology provides a method for selecting a combination of multiple biomarkers for detecting cognitive dysfunction diseases which gives a high percentage of correct answers comprising measuring two or more biomarkers for cognitive dysfunction diseases in a biological sample from a subject, conducting an analysis of the measurement results of each of the aforementioned two or more biomarkers by a logistic regression analysis, and selecting a combination of the biomarkers for detecting cognitive dysfunction diseases which gives a high percentage of correct answers based on the results of the aforementioned analysis.
  • biomarkers and methods for detecting cognitive dysfunction diseases using the biomarkers it is possible to provide biomarkers and methods for detecting cognitive dysfunction diseases using the biomarkers.
  • one or more biomarkers selected from the intact proteins described in (a) to (c) shown below and/or the partial peptides thereof can be used for detecting cognitive dysfunction diseases.
  • the present technology can detect, when a subject has a cognitive dysfunction disease, the type and the level of at least one intact protein and/or partial peptide thereof respectively among those described in the aforementioned (a) to (c) in a biological sample.
  • the present technology enables a diagnosis of whether the subject has a cognitive dysfunction disease or not at a higher accuracy by detecting at least one intact protein and partial peptide thereof among those described in the aforementioned (a) to (c) in a biological sample simultaneously with measuring the change in the type and the level of them.
  • the present technology can provide a system for diagnosis and the like whose accuracy and specificity are both extremely high by measuring one or more biomarkers selected from the aforementioned (a) to (c) in a biological sample. As a result, it is possible to conduct a highly accurate diagnosis and the like with regard to the cognitive dysfunction diseases.
  • the biomarkers of the present technology is highly useful also in judging the efficacy of a drug.
  • the present technology make it possible to, for example, detect, evaluate, discriminate, diagnose, or test that the relevant subject has a cognitive dysfunction disease including mild cognitive impairment or Alzheimer's disease. Furthermore, the present technology allows the diagnosis or the like of whether the subject has a cognitive dysfunction disease or not at a higher accuracy by comparison with a biological sample from a non-demented control subject.
  • two or more of the biomarkers of the aforementioned (a) to (c) are combined to detect the cognitive dysfunction diseases since the accuracy of the detection of the cognitive dysfunction diseases becomes higher.
  • the detection or diagnosis of the cognitive dysfunction diseases can be made at a high accuracy.
  • two biomarkers namely, the aforementioned (c) biomarker (C3) and the aforementioned (a) biomarker (ApoA1) or the aforementioned (b) biomarker (TTR) are measured because the accuracy of the detection of the cognitive dysfunction diseases (MCI and AD) becomes higher.
  • these two biomarkers were detected, there are the cognitive dysfunction diseases (MCI and AD).
  • biomarkers namely, the aforementioned (c) biomarker (C3) and the aforementioned (a) biomarker (ApoA1) when the aforementioned cognitive dysfunction disease detection is the mild cognitive impairment detection, or, to measure the two biomarkers, namely, the aforementioned (c) biomarker (C3) and the aforementioned (b) biomarker (TTR) when the aforementioned cognitive dysfunction disease detection is the Alzheimer's disease detection.
  • peptide of a "partial peptide of an intact protein” of the present technology is meant to include a “polypeptide” and an “oligopeptide”.
  • oligopeptide generally has a molecular weight of 10,000 or less and consists of amino acids bound to each other, or has several to about 50 amino acid residues or less.
  • polypeptide generally has a molecular weight of 10,000 or more and consists of amino acids bound to each other, or has about 50 amino acid residues or more.
  • a partial peptide of an intact protein is a peptide having a partial amino acid sequence which is a part of the amino acid sequence possessed by the intact protein.
  • This partial peptide of an intact protein may sometimes be formed as a partial peptide during expression and synthesis through transcription and translation, or sometimes be formed as a digestion product due to an in vivo digestion after being synthesized as an intact protein. This may be due to deregulation of the mechanism for synthesis and control of the protein in the presence of in vivo condition which is not normal, as in a cognitive dysfunction disease or the like.
  • the present technology enables evaluation, discrimination, and the like of whether a subject is in a normal condition or is having a cognitive dysfunction disease by using an in vivo protein expression and synthesis and/or digestion as an index, and also enables, in case of having the cognitive dysfunction diseases, evaluation, discrimination, and the like of the degree to which the disease has been advanced.
  • detection of cognitive dysfunction diseases means detection of whether a subject has a cognitive dysfunction disease or not, and otherwise it may be evaluation, discrimination, diagnosis or test, and the like.
  • the detection of the cognitive dysfunction diseases of the present technology may include evaluation or the like of the risk at which the subject will have a more serious cognitive dysfunction.
  • the intact protein capable of being employed as a biomarker for detecting cognitive dysfunction diseases may, for example, be apolipoprotein A1 comprising the amino acid sequence represented by SEQ ID NO:1, transthyretin comprising the amino acid sequence represented by SEQ ID NO:2, and complement C3 comprising the amino acid sequence represented by SEQ ID NO:3.
  • a partial peptide of these intact proteins can be employed also as a biomarker for detecting cognitive dysfunction diseases.
  • the partial peptide of an intact protein in the present technology is meant to include the intact protein as well as a peptide fragment having 5 or more amino acid residues generated from a peptide formed during synthesis and degradation thereof.
  • the partial peptide of an intact protein capable of being employed as a biomarker for detecting cognitive dysfunction diseases may, for example, be a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:1 (preferably apolipoprotein A1-derived polypeptide), a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:2 (preferably transthyretin-derived polypeptide), and a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:3 (complement C3-derived polypeptide).
  • SEQ ID NO:1 preferably apolipoprotein A1-derived polypeptide
  • SEQ ID NO:2 preferably transthyretin-derived polypeptide
  • SEQ ID NO:3 complement C3-derived polypeptide
  • the present technology can employ, as a biomarker, a protein or peptide consisting of an amino acid sequence resulting from deletion, substitution, or addition of one or several amino acids in each amino acid sequence of the proteins described in the aforementioned (a) to (c) and partial peptides thereof.
  • a partial peptide employed as a biomarker is meant to include a protein or a peptide comprising the amino acid sequence represented by SEQ ID NOs:1 to 3 as well as peptide fragments generated therefrom having 5 or more amino acid residue.
  • Non-Patent Document 2 reports that a peptide resulting from substituting K for R in the histone H3's C-terminal (130 to 135) amino acid residue sequence IRGERA and a peptide CGGGERA resulting from deletion-of IR and ligation of CGG to GERA were recognized by an antibody obtained by using peptide IRGERA as an immunogen. This reflects that the recognition of the antigenicity is accomplished by a peptide consisting of 4 or more amino acid residues.
  • the number of the residues is 5 or more which is larger by one to ensure a more general coverage rather than limiting to histone H3's C-terminal, it is important to cover even such a low molecular peptide when the detection or separation is conducted by an immunological procedure such as immunoblotting, ELISA, immunoMS method, and the like.
  • a sugar chain may be added to an intact protein or its partial peptide.
  • a protein or its partial peptide to which a sugar chain was added can also be used as a biomarker for detecting cognitive dysfunction diseases.
  • a biomarker may be quantified, or a qualification can be conducted to determine the presence or absence.
  • a biomarker concentration is not lower than a predetermined measured value or when it is not lower than the normal value in the group having no cognitive dysfunction, it is possible to, for example, detect or diagnose that there is a cognitive dysfunction disease.
  • the biomarker quantification it is possible to, for example, detect or diagnose whether the case is positive/negative, and, when there was a reaction with the biomarker and a color was developed or the like, then the case is judged as positive.
  • a two-dimensional electrophoresis or a two-dimensional chromatography can be employed as a method for separating a biomarker in a biological sample such as serum in the present technology.
  • the chromatography employed in the two-dimensional chromatography may be any known chromatography such as ion exchange chromatography, reverse phase chromatography, gel filtration chromatography, and the like.
  • SRM/MRM method employing LC-MS which combines a chromatography (LC) with a triple quadrupole mass spectrometry can be employed for quantification.
  • the LC may be a one-dimensional LC.
  • the present technology can employ, as a method for separating the biomarkers, an immunoMS method (see Patent Document 1) in which beads (including magnetic beads) the inventor developed are allowed to bind to antibodies against target proteins or peptides, thereby capturing the proteins or peptides to be measured which are then allowed to elute from the beads and then measured by mass spectrometry, which enables a convenient evaluation of the existence or the level of the target proteins, protein fragments, or peptides without employing two-dimensional electrophoresis or chromatography.
  • an immunoMS method see Patent Document 1 in which beads (including magnetic beads) the inventor developed are allowed to bind to antibodies against target proteins or peptides, thereby capturing the proteins or peptides to be measured which are then allowed to elute from the beads and then measured by mass spectrometry, which enables a convenient evaluation of the existence or the level of the target proteins, protein fragments, or peptides without employing two-dimensional electrophoresis or chromatography.
  • the type and the level of one or more proteins in a biological sample can be measured simultaneously or separately by various methods.
  • the target protein including protein fragment and its partial peptide
  • the antibody against it primary antibody
  • the present technology preferably involves the measurement conducted by at least one of immunoblotting method, western blotting method, enzyme-, fluorescence-, or radioactive-labeling method, mass spectrometry, immunoMS method, and surface plasmon resonance method.
  • biomarkers of the present technology also enable a simultaneous or separate measurement even when the types or the levels are different.
  • enzyme linked immunosorbent assay ELISA
  • CLIA chemiluminescent immunoassay
  • RIA radioimmunoassay
  • the separated protein is once transferred onto an appropriate membrane such as a PVDF membrane, and a primary antibody and a labeled secondary antibody are employed to conduct a procedure analogous to the aforementioned immunoblotting method, thereby measuring the level of the target protein.
  • an appropriate membrane such as a PVDF membrane
  • a primary antibody and a labeled secondary antibody are employed to conduct a procedure analogous to the aforementioned immunoblotting method, thereby measuring the level of the target protein.
  • An antibody against a protein or its partial peptide is bound to a support such as a microtiter plate which has been modified chemically in a specific manner, and a sample is subjected to a serial dilution, and its appropriate amount is added to the microtiter plate having the antibody bound and then incubated. Thereafter, the protein and the partial peptide which were not captured are washed out. Then, a secondary antibody having a fluorescent or chemiluminescent substance or an enzyme bound thereto is added and incubated.
  • each substrate is added and thereafter the fluorescent or chemiluminescent substance or the enzymatic reaction-induced visible light is measured, thereby accomplishing evaluation and judgment.
  • a substance capable of binding to the protein or its partial peptide may be employed.
  • aptamer can be employed.
  • the present technology preferably employs a substance toward a biomarker described in the aforementioned (a) to (c) (for example, antibody, aptamer, and the like).
  • Patent Document 2 Further methods (see Patent Document 2) are also exemplified below, but they are not limitative.
  • a microarray is a collective name which means a device in which a substance capable of binding to a substance to be measured is immobilized as being aligned (arrayed) on a support (substrate).
  • a support substrate
  • an antibody against the protein or the partial peptide or an aptamer may be aligned and immobilized.
  • a biological sample is added to an immobilized antibody and the like, and a protein or a partial peptide to be measured is bound onto the microarray, and then a secondary antibody having a fluorescent or chemiluminescent substance or enzyme bound thereto is added and incubated.
  • a secondary antibody having a fluorescent or chemiluminescent substance or enzyme bound thereto is added and incubated.
  • each substrate is added and thereafter the fluorescent or chemiluminescent substance or the enzymatic reaction-induced visible light may be measured.
  • an antibody against a certain protein or its partial peptide is bound to a microbead or substrate (protein chip) which has been modified chemically in a specific manner.
  • the microbead may be a magnetic bead.
  • the substrate of any material may be employed.
  • the antibody employed may be all of (1) an antibody which recognizes only the full-length of a certain protein, (2) an antibody which recognizes only a partial peptide, and (3) an antibody which recognizes both of a certain protein and its partial peptide, or a combination of the aforementioned (1) and (2), (1) and (3), or (2) and (3).
  • an appropriate amount of the sample is added as it is or after serial dilution with a buffer solution to a microbead or a substrate to which the antibody has been bound, and incubated. Thereafter, the non-captured protein and partial peptide are washed out. Then, the protein and the partial peptide captured on the microbead or the substrate are analyzed by a mass spectrometry using MALDI-TOF-MS, SELDI-TOF-MS and the like, thereby measuring the peak mass numbers and the peak intensities of the protein, protein fragment, and the partial peptide.
  • a mass spectrometry using MALDI-TOF-MS, SELDI-TOF-MS and the like
  • a sample is separated by means of HPLC as it is or after diluting it with a buffer solution or removing the protein partly, and quantification can be conducted by a mass spectrometry using an electrospray ionization (ESI) method.
  • ESI electrospray ionization
  • the present technology also includes a method for detecting cognitive dysfunction diseases using as an index the existence or the level of the aforementioned biomarker by subjecting a biological sample taken from a subject to a two-dimensional electrophoresis or a surface plasmon resonance.
  • the present technology also includes a device capable of detecting the biomarkers in the aforementioned biological sample (for example, detecting device, measuring device, analyzing device, and the like).
  • the device according to the present technology preferably has an antibody- or aptamer-immobilizing part (capturing part) and a measuring part.
  • the antibody- or aptamer-immobilizing part preferably has a solid support such as a glass slide and 96-well titer plate to which the antibody or aptamer is immobilized.
  • the measuring part preferably has a light detecting means relevant to the detection target, such as a spectrophotometer and fluorescence spectrophotometer.
  • the device of the present technology may include an analyzing part which analyzes the data obtained, and the analyzing part preferably includes a data processing device and a software for analysis.
  • a controlling part including CPU and the like provided in the device disclosed herein or a system capable of being connected thereto is provided with a program capable of conducting the detection, diagnosis, and the like of the cognitive dysfunction diseases of the aforementioned present technology or a memory storing such a program as well as a system.
  • the present technology it is possible to judge a cognitive dysfunction of a subject.
  • the present technology also allows a cognitive dysfunction of a subject to be evaluated at a mild stage, and thus is useful also in preventive medicine. If a mental therapy or a medication given to a patient having a cognitive dysfunction disease serves to suppress the progression of the impairment, the suppression is reflected on the levels of the proteins/partial peptides in biological samples such as serum. By measuring such levels, the effectiveness of the treatment can be evaluated and judged, and screening for a drug discovery target biomolecule becomes possible.
  • the inventor prepared a large number of peptides, to each of which a respective antibody was prepared. Using such an antibody, the biomarkers for detecting the cognitive dysfunction diseases were searched for. For this purpose, an analysis based on a receiver operating characteristic curve (ROC curve) was employed to evaluate the usefulness of each biomarker.
  • ROC curve receiver operating characteristic curve
  • a peptide derived from apolipoprotein A1 consisting of the amino acid sequence represented by SEQ ID NO:1 a peptide derived from transthyretin consisting of the amino acid sequence represented by SEQ ID NO:2, and a peptide derived from complement C3 consisting of the amino acid sequence represented by SEQ ID NO:3 are the markers exhibiting AUCs of 0.6 or more in the analysis based on the receiver operating characteristic curve (ROC curve).
  • the inventor examined whether the multimarker can be used to distinguish the cognitive dysfunction diseases (MCI and AD) from the healthy subject having no cognitive dysfunction diseases or not. For this purpose, a logistic regression analysis was employed in this examination.
  • the present technology can provide a method for selecting a combination of multiple biomarkers for detecting cognitive dysfunction diseases which gives a high percentage of correct answers comprising (i) measuring two or more biomarkers for cognitive dysfunction diseases in a biological sample from a subject, (ii) conducting an analysis of the measurement results of each of the aforementioned two or more biomarkers by a logistic regression analysis, and (iii) selecting a combination of the biomarkers for detecting cognitive dysfunction diseases which gives a high percentage of correct answers based on the results of the aforementioned analysis.
  • MCI or AD is distinguished and the biomarkers for detecting it is selected.
  • ROC curve and the logistic regression curve are as described below in Examples.
  • an optimum biomarker combination can be selected and can serve to raise the percentage of correct answer for the cognitive dysfunction diseases in detection, diagnosis, and the like. Accordingly, by measuring the selected multiple biomarkers, it is possible to conduct a more objective and more accurate detection, diagnosis, and the like for the cognitive dysfunction diseases.
  • the aforementioned (b) biomarker and the aforementioned (c) biomarker are preferably measured simultaneously or separately and used, based on the consequent measurement results, in detection, diagnosis, and the like of the cognitive dysfunction diseases because of the highest percentage of correct answer.
  • the method of the present technology can be stored as a program in a hardware resource provided with a controlling part such as CPU and a recording media (USB memory, HDD, CD, DVD, and the like) and can be executed by the controlling part of a testing device, a selecting device, or the like.
  • a controlling part such as CPU and a recording media (USB memory, HDD, CD, DVD, and the like) and can be executed by the controlling part of a testing device, a selecting device, or the like.
  • the present technology can be utilized also as a cognitive dysfunction disease detection kit using the biomarkers described in the aforementioned (a) to (c).
  • the present technology may also be a cognitive dysfunction disease detection kit for measuring one or more biomarkers for the detecting cognitive dysfunction diseases selected from the aforementioned (a) biomarker (ApoA1), the aforementioned (b) biomarker (TTR), and (c) biomarker (C3).
  • a detection kit combining the (c) biomarker (C3) with the aforementioned (a) biomarker (ApoA1) or the (b) biomarker (TTR) is preferred.
  • the detection kit of the present technology may be a single-component type cognitive dysfunction detection kit containing all reagents capable of detecting the biomarkers described in the aforementioned (a) to (c) or a cognitive dysfunction disease detection kit having multiple (2 or more) detection kits each having a reagent capable of detecting each biomarker in a discrete container.
  • This detection kit preferably includes an antibody against each biomarker of the present technology or an aptamer.
  • one or more biomarkers for detecting cognitive dysfunction diseases selected from the aforementioned (a) biomarker, the aforementioned (b) biomarker, and the aforementioned (c) biomarker in a biological sample are measured simultaneously or separately.
  • a more preferred method for detecting cognitive dysfunction diseases of the present technology comprises (i) measuring two or more biomarkers for detecting cognitive dysfunction diseases selected from the aforementioned (a) biomarker, the aforementioned (b) biomarker, and the aforementioned (c) biomarker in a biological sample from a subject simultaneously or separately, and (ii) judging that the subject has cognitive dysfunction diseases when at least two or more biomarker measurement results are classified to the cognitive dysfunction diseases.
  • the present technology also can employ the following constitution.
  • marker proteins associated to neurodegenerative diseases including Alzheimer's disease
  • those of the complement system namely, complement C3, complement C4, and complement factor H and those involved in suppressing the formation of A ⁇ fibers responsible for cerebral amyloidosis, namely, transthyretin and alpha-2-macroglobulin are employed as markers, and the biological samples obtained from study subjects were examined by an immunoassay method, thereby detecting cognitive dysfunction diseases based on the existence or the level of the marker as an index.
  • a multiplex immunoassay method which simultaneously detects multiple markers (analytes) in the biological samples was employed.
  • Apolipoprotein E (ApoE), apolipoprotein A1 (ApoA1), complement C3 (C3), transthyretin (TTR), complement factor H (Factor H), and alpha-2-macroglobulin (Alpha-2-M) were measured using MILLIPLEX(Trade mark) multiplex kit (HNDG1-36K, Merck Millipore Corporation).
  • Complement C4 (C4) was measured using MILLIPLEX (Trade mark) multiplex kit (HNDG2-36K, Merck Millipore Corporation).
  • the serum samples were employed after 40,000-fold dilution. Thus, 5 ⁇ l of the serum was added to a 1.5 ml tube, to which 995 ⁇ l of the assay buffer was added and stirred gently. A 5 ⁇ l aliquot of this diluted solution was taken into another tube, to which 995 ⁇ l of the assay buffer was added and stirred gently. The resultant solution was used as an assay sample.
  • the serum samples were employed after 2,000-fold dilution. Thus, 5 ⁇ l of the serum was added to a 1.5 ml tube, to which 495 ⁇ l of the assay buffer was added and stirred gently. A 10 ⁇ l aliquot of this diluted solution was taken into another tube, to which 190 ⁇ l of the assay buffer was added and stirred gently. The resultant solution was used as an assay sample.
  • the bead to which an antibody which recognizes the protein to be detected was bound was mixed and diluted to prepare a diluted bead solution.
  • the standard samples for calibration curves were prepared by a serial dilution for 6 concentrations. Thus, 250 ⁇ l of pure water was added to a bottle containing a standard protein, stirred gently and then allowed to stand for 5 to 10 minutes to obtain a standard solution at the highest concentration. A 50 ⁇ l aliquot of this solution was taken into another tube, to which 150 ⁇ l of the assay buffer was added and stirred. Then, 50 ⁇ l of the diluted solution was taken into another tube, to which 150 ⁇ l of the assay buffer was added and stirred. This procedure was repeated 6 times in total. The serial dilutions were 1, 4, 16, 64, 256, 1024, and 4096-fold dilutions.
  • a 96-well assay filter plate for measuring ApoE, ApoA1, C3, TTR, Factor H, and Alpha-2-M and a 96-well assay filter plate for measuring C4 were provided separately, and the antigen-antibody reactions were conducted.
  • the ApoE, ApoA1, C3, TTR, Factor H, and Alpha-2-M measurement plates were subjected to the antigen-antibody reactions at room temperature for 2 hours.
  • the C4 Measurement plate was subjected to the antigen-antibody reaction at 4°C for 16 hours.
  • the solutions were aspirated off, and aspiration was further repeated 3 times while adding 200 ⁇ l of the wash buffer, thereby washing the beads.
  • each 25 ⁇ l of the detection antibodies was added to every well which was employed and then the plate was sealed and allowed to undergo the secondary antibody reaction by stirring at room temperature for 1 hour using the plate shaker.
  • 25 ⁇ l of streptavidin-phycoerythrin was added, sealed, and stirred at room temperature for 30 minutes using the plate shaker.
  • the solution was aspirated off, and aspiration was further repeated 3 times while adding 200 ⁇ l of the wash buffer, thereby washing the beads.
  • Luminex 200 Trade mark
  • xPONENT 3.1 software Luminex Corp., USA was employed and the equation of the standard curve obtained from the standard samples was used for the quantification.
  • AUC of ROC receiver operating characteristic curve
  • Figure 1 , 2 , and 3 show the differential analyses and the ROC curves with regard to the three marker proteins, namely, C3, ApoA1, and TTR.
  • Figure 1 A) shows a differential analysis diagram, and comparison between NDC and MCI indicates MIC exhibiting a significant reduction in C3 (t test, p value: 0.034) and comparison between NDC and AD indicates AD exhibiting a significant reduction in C3 (t test, p value: 1.668E-4). Comparison between MCI and AD also indicates AD exhibiting a significant reduction in C3 (t test, p value: 0.01).
  • Figure 1 B) and Figure 1 C) show the ROC curves of the comparisons of NDC vs. MCI and NDC vs. AD.
  • FIG. 3 A) shows a differential analysis diagram of TTR, and comparison between NDC and AD indicates AD exhibiting a significant reduction in C3 (t test, p value: 5.811E-4).
  • Figure 3 B) and Figure 3 C) show the ROC curves of the comparisons of NDC vs. MCI and NDC vs. AD. The AUC when comparing NDC with MCI was 0.677 while the AUC when comparing NDC with AD was 0.730.
  • Non-Patent Document 3 Czepiel, SA, http://czep.net/stat/mlelr.pdf, 2010, Maximum likelihood estimation of logistic regression models: theory and implementation. This explanation includes the analysis based on Newton-Raphson method.
  • Equations (1) and (2) A large number of data are applied to Equations (1) and (2) to obtain coefficient ⁇ i , and then whose significance (that ⁇ i is not zero) is judged based on the statistic value p.
  • Equation (2) There are two methods for finalizing Equation (2).
  • Equation (2) When a non-significant coefficient was found, then it is excluded to make Equation (2), and the similar application procedure is repeated. When all coefficient of Equation (2) became significant, then, by inserting the measured value of X i , a z value can be obtained from Equation (2). Thereafter, the value of P (discrimination probability) can be obtained from Equation (1). It is also possible to calculate the standard error for the coefficient ⁇ i .
  • the percentage of correct answer is a rate of correct judgment which is the assignment to the group to which the original assignment was made, while, in the method discussed here, all coefficients including coefficients exhibiting no statistical significance are subjected to a trial and error procedure, thereby obtaining the combination giving the highest percentage of correct answer.
  • the discrimination probability is obtained similarly to (I) .
  • the percentage of correct answer of the logistic regression is defined based on the following Equation (3).
  • the odds ratio to a variate x i is a value obtained by dividing the odds of when x i is increased by one unit by the previous odds, and is equal to exp( ⁇ i ).
  • An odds ratio of 1 means that the probability of the occurrence of the event discussed here will not change because the odds is same to the previous one even if increasing x i by one unit.
  • ⁇ i in this case is 0 and there is no contribution to Z.
  • apolipoprotein E apolipoprotein A1
  • C3 complement C3
  • TTR transthyretin
  • Factor H complement factor H
  • Alpha-2-M alpha-2-macroglobulin
  • C4 complement C4

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